The bacterial flagellum is a self-assembling, proton-powered rotary nanomachine that drives motility, allowing chemotaxis. It contains a dedicated type III secretion (T3S) apparatus. Homologous T3S systems are used by pathogenic bacteria to effect virulence, underlying many bacterial diseases such as typhoid fever and bacterial dysentery. Using Salmonella enterica as a model system, we will investigate dynamic interactions among flagellar proteins that allow membrane translocation of export substrates through use of molecular biology, genetics and biochemical techniques including optical biosensing. The specific aims are to complete a kinetic survey of interactions among export apparatus proteins using optical biosensing as the primary tool, to examine higher-than-pairwise interactions that better reflect in vivo dynamics and to further the development of an in vitro T3S assay. Eventual development of an in vitro export assay will be of tremendous significance because it would allow us to examine hypotheses addressing T3SS function currently out of reach. Since the flagellar export apparatus is highly similar to virulence T3S systems, the project will contribute significantly to our understanding of bacterial pathogenesis. AREA program goals of enhancing the research environment and exposing students to meritorious research will be well-served by this project.